Significant progress during the current reporting period has occurred in four areas: (1) publication of a manuscript describing three lines of mice that are knockins for human mutations causing FMF, which provide new insight into the mechanism of this disease as well as the regulation of inflammation more generally;(2) in collaboration with Dr. Phil Hashes, now in Jerusalem, completion of a clinical trial testing the efficacy of rilonacept, a long-acting interleukin-1 blocker, in patients with FMF who are unresponsive to or intolerant of the standard treatment, colchicine;(3) in collaboration with Dr. Kerstin Linblad-Toh, publication of a paper describing the molecular basis of a spontaneous FMF-like illness in the Shar-Pei dog;and (4) in collaboration with Dr. Raphaela Goldbach-Mansky of NIAMS, discovery of a new recessively inherited autoinflammatory disease characterized by chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE syndrome). Pyrin knockin mice To study the role of FMF-associated mutations in the C-terminal B30.2 domain of human pyrin in the molecular pathogenesis of disease, we generated KI mouse models by inserting the B30.2 domain of wild-type (WT) or FMF-associated M680I, M694V, and V726A mutant pyrin into mouse pyrin, which ordinarily does not include a B30.2 domain (although there is strong homology with human pyrin at the nucleotide level at the 3 prime end of the gene). While the WT human B30.2 was embryonic lethal, the mice homozygous for human FMF mutations exhibited a phenotype similar to human disease, but that was chronic rather than episodic, and generally more severe. KI mice exhibited growth retardation, spontaneous dermatitis and arthritis, and increased CD11b+ cells (especially Ly-6G+ neutrophils) in the blood. Somewhat surprisingly, the phenotypic severity in the mouse was inversely proportionate to the severity of the mutations in humans, with M694V the most severe mutation in humans but the least severe in the mouse, and V726A the least severe mutation in humans but the most severe in the mouse. The inflammatory phenotype was observed in homozygous KI mice, but not heterozygous or hemizygous mice, suggesting that FMF-associated mutations are gain-of-function, with a gene-dosage effect. Bone marrow (BM) cells of KI mice transferred the phenotype into WT mice, and WT BM cells rescued the diseased KI mice, suggesting that BM-derived cells are necessary and sufficient for the disease. Lymphocytes are not required for inflammation since Rag-1 deficient KI mice showed phenotypes similar to Rag-1 sufficient KI mice. In CD45 congenic mixed bone marrow chimeras we found evidence that KI hematopoietic cells could induce WT CD11b expansion. Proinflammatory cytokines, such as IL-1beta, were significantly increased in the KI mouse sera. Caspase-1 was constitutively activated in KI macrophages and active IL-1 beta was secreted when stimulated with LPS alone, without ATP, which is also observed in FMF patients. The inflammatory phenotype of KI mice was completely ablated by crossing with IL-1 receptor KO or Asc KO mice, but not Nlrp3 KO mice. Taken together, these data provide evidence for a heretofore-unrecognized ASC-dependent NLRP3-independent inflammasome in which gain-of-function mutations cause autoinflammatory disease. This work was published earlier this year in Immunity. Rilonacept in FMF Currently there is no proven alternative for FMF patients resistant to or intolerant of colchicine. We assessed the efficacy and safety of rilonacept, an interleukin-1 decoy receptor, using a double-blind, single-subject alternating treatment design. Subjects over 4 years old with at least 1 monthly attack despite adequate doses of colchicine, or who were colchicine intolerant, were recruited at 6 U.S. sites. Subjects were randomized to 1 of 4 treatment sequences that included two 3-month courses of rilonacept 2.2 mg/kg (max 160 mg) by weekly SC injection, and two 3-month courses of placebo. Eight males and 6 females were randomized with a mean age (standard deviation) of 24.4 (11.8) years and 3.1 (2.0) attacks per month at baseline. Among 12 subjects who completed at least 2 treatment courses, the primary outcome, the rilonacept/placebo risk ratio for attacks by Bayesian analysis (consistent for all prior assumptions) was 0.57 (0.17) (0.31-0.98 posterior 2.5-97.5 percentiles). The mean number of attacks per month was 1.0 (1.2) on rilonacept vs. 1.8 (0.9) on placebo (P = 0.027). There were significantly more courses of rilonacept without attacks (P = 0.004) and with a greater than 50% decrease in attacks compared to baseline than with placebo courses (P = 0.006). There were 2 respiratory infection serious adverse events, 1 on rilonacept and 1 on placebo. Injection site reactions were significantly more frequent with rilonacept (P = 0.039) but no differences were seen in other adverse events. We concluded that rilonacept significantly reduces the frequency of FMF attacks and has an acceptable safety profile, appearing to be a treatment option for colchicine resistant or intolerant patients. A manuscript describing these findings is nearly ready for submission. Shar-Pei fever A hereditary periodic fever syndrome similar to FMF is relatively frequent in the Shar-Pei breed of dogs. In the western world, Shar-Pei have been strongly selected for a distinctive thick and heavily folded skin. In this study, a mutation affecting both of these traits was identified. Using genome-wide SNP analysis of Shar-Pei and other breeds, the strongest signal of breed-specific selective sweep was located on chromosome 13. The same region also harbored the strongest genome-wide association signal for susceptibility to the periodic fever syndrome (P genome = 0.01). Dense targeted resequencing revealed two partially overlapping duplications, 14.3 Kb and 16.1 Kb in size, unique to Shar-Pei and upstream of the Hyaluronic Acid Synthase 2 (HAS2) gene. HAS2 encodes the rate-limiting enzyme synthesizing hyaluronan (HA), a major component of the skin. HA is up-regulated and accumulates in the thickened skin of Shar-Pei. A high copy number of the 16.1 Kb duplication was associated with an increased expression of HAS2 as well as with the periodic fever syndrome (P less than ten to the negative fourth). When fragmented, HA can act as a trigger of the innate immune system and trigger sterile fever and inflammation. The strong selection for the skin phenotype appears to enrich for a pleiotropic mutation predisposing these dogs to a periodic fever syndrome. The identification of HA as a major risk factor for this canine disease raises the potential of this glycosaminoglycan as a major risk factor for human periodic fevers and as an important driver of chronic inflammation. This work was published earlier this year in PLoS Genetics. Genetic basis of a new autoinflammatory disease CANDLE syndrome is characterized by onset during the first year of life, recurrent fevers, purpuric skin lesions, arthralgia, progressive lipodystrophy, anemia, and delayed physical development. Four of 8 CANDLE patients screened in our laboratory were homozygous for the T75M mutation in PSMB8, a gene encoding a component of the immunoproteosome that has recently been implicated in two other clinical forms of lipodystrophy. One of the remaining 4 patients was heterozygous for the T75M PSMB8 mutation, and bore a heterozygous 3 bp in-frame deletion for PSMA3, another proteasome component. A sixth CANDLE patient was compound heterozygous for mutations in PSMB4, encoding yet another proteasome component. These data suggest that the phenotype of panniculitis and lipodystrophy may be caused by several different lesions in the proteasome complex. A manuscript describing this work is in press in Arthritis and Rheumatism.